-->PONE-D-24-43253-->-->A new species of Brachycephalus (Anura: Brachycephalidae) from Serra do Quiriri, northeastern Santa Catarina state, southern Brazil, with a review of the diagnosis among species of the B. pernix group and conservation measures-->-->PLOS ONE

Dear Dr. Pie,

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We look forward to receiving your revised manuscript.

Kind regards,

Neelesh Dahanukar, Ph.D.

Academic Editor

PLOS ONE

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“The field work was funded by Fundação Grupo Boticário de Proteção à Natureza (through grant 1149_20191) through project conducted by Mater Natura – Instituto de Estudos Ambientais. MRP received a grant from CNPq/MCT (301636/2016-8). GS-S received  grant from São Paulo Research Foundation (FAPESP; processes #2022/04847-7 and # 2023/09718-3). There was no additional external funding received for this study.”

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Additional Editor Comments:

There are several issues with the manuscript that authors will have to address before the manuscript can be considered further for review.

(1) Defining the Brachycephalus pernix group: Authors mention the B. pernix group in the introduction without properly defining it and providing information about the number of species under this group. The references used for defining the group are old and several species have been published ever since. So there is a need to provide the number of species currently present in the group and also list them for easy reference to the readers. Based on Table 2, it seems that there are 20 species currently know in this species group. If this is the case, why does the number of species differ substantially for different types of analysis and comparison. For example, the genetic analysis is based on only six species, while the conservation analysis is for 12 species. Authors need to keep some consistency in the arguments and explain why other species are not considered for the given analysis.

(2) Phylogenetic analysis: Phylogenetic analysis is not presented properly. Authors are not allowed to pick and choose some species for phylogenetic analysis. Molecular data are available for several species of B. pernix group and authors should use all available species in the group for analysis, even if some of the genetic markers are missing. In fact, it is advisable that authors use all member so the genus for larger phylogeny to show the monophyly of the group and then the taxonomic position of the new species. The genetic analysis is also not presented properly. The outgroups are not defined. There are large branches within the clades and there are politomies, suggesting that the trees are not resolved. Authors should increase the taxonomic sampling and perform maximum likelihood analysis in addition to Bayesian analysis. The genetic distances provided by the authors make very little sense. If there are multiple individuals then there will be a range of genetic distance and not just one value. It is essential to provide this range (minimum-maximum) for the intra and inter species genetic distance. I will refrain from providing the mean as the number of individuals are low and even if means are provided they should be accompanied with the standard deviation or some measure of uncertainty. The long branches in the proposed species are worrisome, and it is essential that authors prove that the new species is genetically distinct with species delimitation methods such as barcode gap (using ASAP) and Poisson tree process.

(3) Conservation status: This is the weakest part of the manuscript. Authors are using methodology that they themselves have devised for defining IUCN categories and they are not referring to proper IUCN guidelines for defining the categories based on the various criteria that are defined by the IUCN Redlist of Threatened Taxa documentation. If authors are using their own methodologies, there is no point in using the IUCN categories for defining the conservation status. The IUCN guidelines provides proper rationale for when to use different categories based on five different criteria used for assessments. Author's argument that the species is DD as a polygon cannot be constructed is flawed. The point localities are enough to define the Area of Occupancy based on 2km grids and have been used for defining the conservation status where the species are point endemics. Either authors should understand how the various criteria are used for finding the IUCN categories for red listing or authors should not used the categories used by the IUCN. In the current case if the threats to the species are know and the distribution is known, the species is not DD.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Partly

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

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-->3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

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Reviewer #1: No

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: This manuscript proposes a new species of the anuran genus Brachycephalus . The authors present several lines of evidence, including external morphology, osteology, vocalizations and molecular data to support the status of this taxon as a new species. They also acknowledge the difficulty in differentiating many species of the B. pernix group due to their morphological similarity and/or shallow genetic distances and highlight the importance of using as many types of data as possible in species descriptions.

However, I think the manuscript has a number of problems that should be addressed before it can be considered for publication, including minor issues such as wording, grammar, sentence structure, etc.

The complete review and comments on the ms are uploaded here as a file, due to its size.

**********

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Reviewer #1: No

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Attachments

Attachment

Submitted filename: PONE-D-24-43253_review.docx

-->PONE-D-24-43253R1-->-->A new species of Brachycephalus (Anura: Brachycephalidae) from Serra do Quiriri, northeastern Santa Catarina state, southern Brazil, with a review of the diagnosis among species of the B. pernix group and conservation measures-->-->PLOS ONE

Dear Dr. Pie,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.-->--> -->-->In general the comments raised on the earlier draft by both the reviewer and the academic editor are addressed by the authors. However, the reviewer has provided some more comments, including reiteration of some of the comments made on the earlier draft. It is advisable that authors take all the comments made on the earlier and the current draft seriously and make appropriate changes to the manuscript. Authors should also provide a point to point rebuttal to the concerns raised by the reviewer. 

Please submit your revised manuscript by Sep 20 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:-->

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

-->If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Neelesh Dahanukar, Ph.D.

Academic Editor

PLOS ONE

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If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 

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Additional Editor Comments:

Authors have revised the text substantially with respect to the comments provided by the reviewer and the academic editor. However, the reviewer has provided some more comments, including reiterating earlier comments, and it is advisable that authors should take these comments seriously and provide proper justification to the issues raised and include the justification even int he main text of the article.

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Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #1: (No Response)

**********

-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

**********

-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

**********

-->4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

**********

-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

**********

-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: Major issues:

As stated in my first review of this ms, I still discourage the continuing use of the B. didactylus group as a taxonomic entity. The authors have responded to this critic by arguing that the B. didactylus group is “a very well-defined group of species based on morphological characteristics”. Species groups are treated in current zoological systematics as monophyletic groupings of species rather than purely phenetic ones (unless when the phylogenetic relationships among species are not known). And it is now widely known that the characteristics uniting the species in the B. didactylus group (including B. clarissae, which does not share all of them) are plesiomorphic for the genus. Yet, even after addressing the non-monophyly of the B. didactylus group in the introduction, the authors continue to refer to it throughout the text as if it was a taxonomic entity equivalent to the other two species groups in the genus (for which there is strong evidence of monophyly in the literature). I consider this misleading, and continue to advocate for the use of the term “flea toads” instead, which unlike the “species group” category, corresponds to a morphotype rather than a clade. Indeed, the term is adopted in the work of Toledo et al. (2024), which included B. clarissae among the species with that morphotype (as mentioned by the authors in their response to my previous review).

The authors also seem to use the B. ephippium group in the sense of Ribeiro et al. 2015 (i.e. including both the ephippium and vertebralis lineages proposed by Condez et al. 2020) rather than in the sense of Folly et al. 2022 (i.e. excluding the vertebralis lineage, which is considered as a separate species group). The latter definition seems more conservative, since not all phylogenetic analyses published so far presented strong support for the monophyly of the B. ephippium group sensu Ribeiro et al. 2015. Anyway, since there are currently two different definitions of this group being used in the literature, the authors should explicitly state in the text which one they are using.

I believe that in all tables, as well as in table and figure legends that feature the name “B. lulai”, it should be followed by “sp. nov.”

Minor corrections:

Line 4 – I suggest adding the word “proposed” before “conservation measures”.

Line 26 – Exclude the hyphen in “currently-recognized”.

Line 29 – Change “call comparisons” to “acoustic comparisons”.

Line 52 – “The species are diurnal”. This should be changed to “…mostly diurnal”, since some of the cryptically colored “flea toads” often present nocturnal activity.

Line 98 – Change “also inhabited de occurrence of” to “also inhabited by”.

Line 102 – Change “occurs B. leopardus, a species known from two localities” to “B. leopardus occurs, being known from two localities”.

Lines 106-107 – Change “annual mean of temperature” to “mean annual temperature”.

Line 306 – Change “we did not mapped” to “we have not mapped”.

Line 468 – “discrete irregular greenish or brown spots”. Do you mean “discrete” or “discreet”? These are different terms.

Line 479 – Add the word “species” after “Sexually dimorphic”.

Line 483 – Change “green spots” to “green patches”.

Line 486 – Change “becoming dark, brownish black” to “becoming dark gray”.

Line 549 – “in B and C, von Kossa staining”. I believe there is an error here: it should be “B and D”, right?

Line 643 – Change “in the southeastern hillside from” to “on the southeastern hillside of”.

Line 645 – Change “it is likely that the B. lulai populations” to “it is likely that B. lulai populations”.

Line 652 – Exclude the word “with”.

Lines 662-663 – Change “of B. lulai” to “of one individual of B. lulai”.

Lines 664-665 – Change “previously studied parasites [58] and found under the same conditions” to “previously recorded parasites found under similar conditions [58]”.

Line 665 – Change “without apparent morphological structures and in a young stage” to “without apparent diagnostic morphological structures and at a young stage”.

Lines 667 – Add the word “presumably” before “makes”.

Lines 668-670 – This sentence starting with “Furthermore…” is confusing and it is not clear what it is trying to say. I suggest deleting this sentence and replacing it with this: “Indeed, Ophiotaenia spp. are generalist parasites that use not only amphibians, but also snakes and turtles as hosts [58].”

Line 671 – Change “the Ophiotaenia parasite was found in” to “Ophiotaenia parasites have been found in”.

Lines 671-672 – I think the record of Ophiotaenia in B. pernix by Ribeiro et al. (2014) should be cited here too.

Line 676 – Change “of the parasite at the gular region” to “of the parasites in the gular region”.

Line 692 – Change “even with the small distribution.” To “even considering its small distribution area.”

Lines 693 and 699-700 – Change “From the 20 remaining species” to “Of the 20 remaining species”.

Line 706 – Add the word “states” after “Santa Catarina”.

Table 7 – In the line of B. pombali, change 1300 to 1,300.

Line 735 – After “digital reduction”, I suggest adding “(President Lula lost the little finger on his left hand in an accident)”. I think an explanation is appropriate, since this pun may not be obvious for some non-Brazilian readers.

Line 749 – Change “support our interpretation” to “reinforce our interpretation”.

Line 759 – Change “vertebrae” to “vertebra”.

Lines 783 and 784 - Change “other words” to “other works”.

Line 784 – Change “described to as” to “described as”.

Line 819 – Change “did not note this feature” to “did not mention this feature”.

Line 825 – Change “latter it was demonstrated” to “it was later demonstrated”.

Line 862 – Change “would be as an example” to “would be an example”.

Line 883 – Change “expanding the knowledge of intraspecific variation” to “the expanding knowledge on intraspecific variation”.

Line 907 – Change “increasingly” to “increasing”.

Line 907 – Change “which we have sometimes achieved” to “which is sometimes achieved”.

Line 926 – Change “mountain top” to “mountaintop”.

Line 932 – Change “vegetative front” to “arboreal stratum”.

Line 964 – Change “Brachycephalus ’s geographic range” to “the geographic range of species in the genus Brachycephalus ”.

Line 970 – Change “propose” to “hypothesize”.

Line 971 – Change “upslope” to “upward”.

Line 976 – Change “forest” to “vegetation”.

Line 983 – Add “to” after “according”.

Line 986 – Change “to Santa Catarina” to “to the state of Santa Catarina”.

Line 999 – Change “possibly” to “possible”.

Line 1000 – Change “conservation target species and key areas” to “target species and key areas for conservation”

Line 1034 – Change “and, mainly, take” to “and, especially, change the status of”.

Line 1035 – Delete “the”.

Line 1036 – Add the word “anthropic” before “impacts”.

Line 1043 -Change “and confirm” to “to confirm”.

Line 1069 – Add “may have” before “occurred”.

Line 1079 – “the lacks of” to “the apparent lack of”.

Line 1092 - Change “improve” to “improved”.

Line 1383 – Put the name “Pleroma” in italics.

**********

-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .-->

Reviewer #1: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.-->

A new species of Brachycephalus (Anura: Brachycephalidae) from Serra do Quiriri, northeastern Santa Catarina state, southern Brazil, with a review of the diagnosis among species of the B. pernix group and proposed conservation measures

PONE-D-24-43253R2

Dear Dr. Pie,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Neelesh Dahanukar, Ph.D.

Academic Editor

PLOS ONE